Kasetsart J. (Nat. Sci.) 43 : 358 - 369 (2009)

Antioxidant Activities and Antioxidative Components in Extracts of Alpinia galanga (L.) Sw.

Nopparat Mahae and Siree Chaiseri*

ABSTRACT

Galangal extracted using 50% ethanol in water was studied for its antioxidant activity and composition in comparison with two other samples based on a water extract and the essential oil. The antioxidant activities were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) methods. The ethanolic extract showed the highest DPPH free radical scavenging ability as well as the highest ORAC value when compared to the water extract and the essential oil. The IC50 values of the galangal ethanolic extract (10.66 mg/ml), water extract (55.48 mg/ml) and essential oil (455.43 mg/ml) were higher than those of α-tocopherol (1.45 mg/ml) and butylated hydroxyanisole (BHA; 0.41 mg/ml). The results indicated that the antioxidant activities of galangal extracts were lower than that of BHA, the commercial synthetic antioxidant generally used in food. The ethanolic extract contained the highest concentrations of total phenolic compounds (31.49 mgGAE/g) and flavonoids (13.78 mgCE/g). The water extract and the essential oil had a total phenolic content of 8.25 and 5.01 mgGAE/g and a total flavonoid content of 1.48 and 0.20 mgCE/g, respectively. Antioxidants in the galangal essential oil, namely, methyl eugenol (4,130.38 µg/g), chavicol (2,390.45 µg/g), and eugenol (728.30 µg/g) were found in its volatile fraction. The water extract contained mainly myricetin (14.60 mg/g extract) and an unknown phenolic compound. The major antioxidants in the ethanolic extract were 1´-acetoxycavichol acetate (10.56 mg/g extract), catechin (1.74 mg/g extract), and three unknown subtances. In addition to the phenolic compounds, 1´-acetoxycavichol acetate (ACA) could play an important role in the antioxidant activity of galangal. The ethanolic extract of galangal was also advantageous as an antioxidant in food due to its mild odor compared with the essential oil. Key words: Alpinia galanga, galangal extract, antioxidants, essential oil, phenolic compounds, 1´-acetoxycavichol acetate

INTRODUCTION medicine (Yang and Eilerman, 1999), as the essential oil shows an antimicrobial activity Galangal (Alpinia galanga) is used to against gram-positive bacteria, yeast, and flavor foods throughout South and Southeast Asia. dermatophytes. The most active compound is Its aromatic characteristic is described as woody, terpinen-4-ol (Janssen and Scheffer, 1985). In minty and floral (Mori et al., 1995). Its rhizome addition, essential oil from galangal was reported has a wide range of applications in traditional as a potential anti-carcinogen (Zheng et al., 1993).

Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand. * Corresponding author, e-mail: [email protected] Received date : 15/09/08 Accepted date : 07/01/09 Kasetsart J. (Nat. Sci.) 43(2) 359

Several researches reported that 1´- extract with regard to its antioxidant activity in acetoxychavicol acetate (ACA) and 1´- comparison with a water extract and the essential acetoxyeugenol acetate from galangal were anti- oil. tumor substances (Itokawa et al., 1987; Kondo et al., 1993). MATERIALS AND METHODS Natural antioxidants from plants have attracted significant interest because of their safety Chemicals and apparatus and potential nutritional and therapeutic effects. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Several plant materials have been investigated as fluorescein sodium salt (FL) and (+)-α-tocopherol a potent source of antioxidants. Antioxidants in were purchased from Sigma-Aldrich (Milwaukee, herbs and spices include: vitamins; phenolic WI, USA). 6-Hydroxy-2,5,7,8-tetramethyl- compounds including flavonoids and phenolic chroman-2-carboxylic acid (Trolox) and 2,2´- acids; and volatile compounds (Carrubba and azobis (2-methylpropionamidine) dihydrochloride Calabrese, 1998). The antioxidant activity of (AAPH) were obtained from Sigma-Aldrich phenolic compounds is mainly dependent on their (Munich, Germany), while the gallic acid was redox properties that allow them to act as reducing sourced from Fluka (Madrid, Spain) and Folin- agents, hydrogen donators and singlet oxygen Ciocalteu’s phenol reagent from Fluka (Buchs, quenchers. Additionally, they have a metal- Switzerland). Randomly methylated beta chelating potential (Rice-Evans et al., 1995). cyclodextrin (RMCD) for the ORAC test was Several studies have been conducted on the purchased from Cyclodextrin Technologies antioxidant activities of galangal extracts. The Development (Gainesville, FL, USA). Acetone, antioxidant activity of a 1% (w/v) acetone extract ethanol, and methanol came from Merck of galangal in 99.5% ethanol was stronger than (Darmstadt, Germany). A fluorescent microplate that of α-tocopherol (Jitoe et al., 1992). Zaeoung reader (FLUOstar OPTIMA, BMG Labtech, et al. (2005) found the new phenolic compound, Offenburg, Germany) and fluorescence filters with p-coumaryl-9-methyl ether, in the methanolic an excitation wavelength of 485 nm and emission extract of fresh galangal. Moreover, two phenolic wavelength of 520 were used. The 96-well black compounds, p-hydroxy cinnamaldehyde and [di- microplates were purchased from BMG Labtech. (p-hydroxy-cis-styryl)] methane, were isolated Volatile compounds were isolated with a high from the chloroform extract of dry galangal (Barik vacuum distillation unit (Edwards, West Sussex, et al., 1987). In food systems, research has mostly UK) and a spectrophotometer (Spectro 23, been carried out on the ethanolic extract (Cheah LaboMed, Inc., USA) was used for the and Abu Hasim, 2000; Juntachote et al., 2006). spectrophotometric assay. The ethanolic extract of galangal acted as a free radical scavenger, exhibiting strong superoxide, Sample preparation and extraction anion-scavenging activity, Fe2+ chelating activity Fresh rhizomes of galangal were and reducing power in a concentration-dependent purchased from local markets in Bangkok and manner (Juntachote and Berghofer, 2005). cleaned with tap water. The extracts were prepared Although, there were several studies on the from fresh and dried galangal. Dried galangal was antioxidant activities of galangal ethanolic prepared by freeze-drying the fresh rhizomes using extracts, the information related to their a freeze dryer (Heto Lab Equipment, Allerod, composition was limited. This study aimed to Denmark). The dried sample was ground to a fine investigate the composition of galangal ethanolic powder using a blender and then passed through a 360 Kasetsart J. (Nat. Sci.) 43(2)

60-mesh sieve. Antioxidant activity Three types of the extracts were The antioxidant activity of the water prepared: ethanolic extract, water extract and extract, ethanolic extract and essential oil from the essential oil. The ethanolic extract and water galangal rhizome were studied using DPPH and extract were prepared from galangal powder ORAC methods. The use of galangal extracts whereas the essential oil was steamed-distilled together with BHA in methanol was also from fresh galangal. investigated by the DPPH method to study the effects of using the extract with a commercial Ethanolic extract antioxidant. A comparative study on the Galangal powder was extracted using antioxidant activities of the samples, BHA and 50% ethanol (v/v) in water at a solid-to-solvent α-tocopherol was also conducted using the DPPH ratio of 1:10. The extraction was performed in a method and reported as IC50 . shaking water bath at 50°C for 1 h. The extract was filtered through a No.1 sinter glass funnel. DPPH assay The residue was re-extracted using the same The electron-donation abilities of the conditions. The two extracts were then combined extracts and commercial antioxidants were and concentrated in a rotary evaporator at 40°C. measured from the bleaching of purple DPPH in The concentrated extract was dried using a freeze methanol solution. dryer and kept in a glass vial at –40°C. The DPPH assay was carried out as described by Tepe et al. (2005). The ethanolic Water extract extract and the water extract were diluted with Ten grams of galangal powder were water and the essential oil was diluted with ethanol refluxed with 100 ml of distilled water for 3 h at to various concentrations. The concentrations of 80°C. The mixture was cooled to room temperature the ethanolic extract were from 0 to1.0% (w/v), and filtered through a No.1 sinter glass funnel. The the water extract samples were from 0 to 5.0% residue was then re-refluxed with 100 ml distilled (w/v), while the essential oil sample ranged from water under the same conditions. The extracts were 0 to 25% (w/v). The DPPH studies were conducted combined and concentrated in a rotary evaporator with and without the addition of BHA. The at 60°C. The concentrated extract was freeze dried ethanolic extract and water extract samples were and kept in a glass vial at – 40°C. supplemented with 0.01% BHA (w/v) in methanol, whereas the essential oil samples were Essential oil supplemented with 0.02% BHA (w/v) in methanol, The essential oil was obtained from fresh in order to be able to observe the antioxidant galangal rhizomes by steam distillation using a activities. glass essential oil extractor (Becthai Bangkok In the DPPH assay, 50 µl of samples at Equipment and Chemical, Bangkok, Thailand). various concentrations were added to 5 ml of 0.004 The fresh rhizomes were cut into small pieces % DPPH in methanol. After a 30 min incubation (1 cm × 1 cm × 0.3 cm) and 320 g of galangal was period at room temperature, the absorbance was comminuted with 400 ml water. The slurry was read at 517 nm. The purple color bleaching of the prepared in a one-liter, round-bottomed flask and DPPH reagent was evaluated as positive steam-distilled for 4 h. The essential oil was dried antioxidant activity. The inhibition of free radicals by filtering through anhydrous sodium sulfate and by DPPH in percent (I %) was calculated using kept in a glass vial at – 40°C. Equation 1: Kasetsart J. (Nat. Sci.) 43(2) 361

I %=[(AB-AA)/AB] × 100 (1) (2003). A volume of 0.25 ml of a known dilution where: extract (5%, 1% and 12.5% for the water extract,

AB= the absorbance of a blank after 30 ethanolic extract and essential oil, respectively) min, and and 1.25 ml of distilled water were added to a test

AA= the absorbance of the sample after tube. Then, 0.075 ml of 5% sodium nitrite solution 30 min. was added to the mixture. The reaction was For the comparative study on the allowed to progress for 5 min. After that, 0.15 ml antioxidant activities of the samples, BHA and a- of 10 % aluminum chloride was added. After 6 tocopherol, the results were reported as IC50 (the min, 0.5 ml of 1 M sodium hydroxide was added concentration of an antioxidant to achieve 50% and the mixture was diluted with 0.275 ml of inhibition of free radicals by DPPH assay). distilled water. The absorbance at 510 nm was read immediately. A calibration curve was prepared

ORACFL method with (+)-catechin. The flavonoid content was The ORAC assay in this study followed expressed as mg (+)-catechin equivalent (CE)/g the method using fluorescence described by Prior sample. et al. (2003). The antioxidant activity was quantified by calculating the net protection area High performance liquid chromatography under the time-resolved fluorescence decay curve. (HPLC) analysis

AAPH, a peroxyl radical generator (ORACROO.), Identification and quantification of the was used. The data were expressed as an ORAC non-volatile phenolic compounds were achieved value (µmol Trolox equivalent (TE)/g extract). using an Agilent 1100 HPLC Series system. The ethanolic extract was dissolved in methanol and Phenolic compound analysis filtered through a 0.45 µm regenerated cellulose Determination of total phenolic compounds membrane filter. Samples were separated on a The total phenolic content of the galangal reversed-phase Zorbax SB-C18 analytical column extracts was determined by a modification of the (100 × 3.0 nm i.d., 3.5 µm particle). The column Folin-Ciocalteu method, according to Wolfe et al. was operated at 48°C. A variable UV-VIS detector (2003). Folin-Ciocalteu reagent (0.125 ml) was was used to detect the compounds at 280 nm. The added to a test tube containing 0.5 ml of deionized gradient mobile phases were methanol and a water and 0.125 ml of a known dilution of the potassium dihydrogen phosphate buffer solution. extract. The reaction was allowed to progress for The buffer solution was prepared by dissolving 6 min, then 1.25 ml of 7% sodium carbonate potassium dihydrogen phosphate (40 mM) in solution was added to the test tube and the mixture deionized water and adjusting the pH to 2.3 with was diluted to 3 ml with deionized water. The 85% orthophosphoric acid. The binary gradient mixture was allowed to stand for 90 min. The started with a linear gradient from 5% to 42% absorbance was then measured at 760 nm. The methanol in the first 35 min, followed by isocratic content of the phenolic compounds was expressed elution with 42% methanol for the next 3 min. The as mg gallic acid equivalent (GAE)/g sample. flow rate was 1 ml/min and the injection volume was 10 µl. The solutions were filtered through a Determination of flavonoid content 0.45 mm nylon membrane filter and degassed in The determination of the flavonoid an ultrasonic bath before use. Retention times and content was performed according to the spectra were compared to those of the pure colorimetric assay described by Wolfe et al. standards. 362 Kasetsart J. (Nat. Sci.) 43(2)

Gas chromatography-mass spectrometry (GC- temperature program, which commenced with an MS) analysis initial temperature of 40°C heated to 210°C at a For the ethanolic and water extracts, 1 g rate of 10°C/ min and held for 30 min. of the extract was dissolved in 25 ml of deodorized For the essential oil, the analysis was water in a 250 ml glass bottle. The sample was modified due to the differences in solubility and then supplemented with 20 µl of the internal the profile of the volatile compounds in the sample. standard (10 µg/ml of 2-undecanol in One microliter of diluted essential oil was injected dichloromethane). The solution was consecutively with the split ratio of 1:5. Then 2-Undecanol extracted with 25 ml dichloromethane on a stirring solution (10 mg/ml dichloromethane), an internal plate for 30 min. The extract was then transferred standard, was added to the sample at the to a 250 ml round-bottom flask, which was concentration of 10 µl/ml. The HP-5 capillary immersed in liquid nitrogen until the extract was column was used under the conditions previously frozen. Volatile compounds and the solvent were described. The HP-FFAP was used under the vacuum-distilled at 10-5 Torr at room temperature following conditions: split-splitless inlet, 200°C; for 1 h and at 50°C in a water bath for 1 h. The detector temperature, 280°C; and an oven volatile extract was recovered from the first temperature program with an initial temperature receiving tube and concentrated to 5 ml under a of 40°C, heated to 210°C at a rate of 6°C/ min and gentle stream of nitrogen. The concentrated extract held for 10 min. Helium was used as the carrier was then dried over anhydrous sodium sulfate and gas at the flow rate of 1.3 ml/min. then further concentrated to 1 ml under a gentle Compounds were identified using stream of nitrogen. The sample was stored in a authentic standards, matching mass spectra with glass vial at -40°C until used for analysis. For the the Wiley275 Mass Spectra Database and by essential oil sample, the oil was diluted to 1/100 comparison of the retention indices (RIs) related (v/v) in dichloromethane. to the retention times of the n-alkane series. Qualitative and quantitative analyses of Relative concentrations were determined using the the extracts were performed using an HP 6890 gas MS response factors for each component related chromatograph equipped with an HP 5973 mass to the internal standard. selective detector (MSD, Agilent Technologies, Palo Alto, USA). The MSD was operated at Statistical analysis 70 eV, with a scanning speed of 1 s over a 40-300 The data were subjected to analysis of amu range with an ion-source temperature of variance (ANOVA). The significance of the 180°C. An HP-5 and an HP-FFAP column were difference between means was determined by used for separation of the volatile compounds. The Duncan’s multiple-range test (p ≤ 0.05) using HP-5 capillary column (60 m × 0.25 mm × 0.25 SPSS version 12 (SPSS Inc., Chicago, IL). The µm) was used under the following conditions: inlet correlations between the phenolic content, temperature, 200°C; detector temperature, 280°C; flavonoid concentrations and antioxidant activity ans an oven temperature program with an initial were performed using Pearson’s correlation temperature of 35°C heated to 250°C at a rate of coefficient. For GC and HPLC analysis, the 10°C/ min and held for 10 min. Helium was used experiments were carried out in duplicate. The as the carrier gas at the flow rate of 1.5 ml/min. other experiments were performed in triplicate. The split ratio was 1:5. The HP-FFAP capillary column (60 m × 0.25 mm × 0.25 µm) was used under the same GC conditions, except for the oven Kasetsart J. (Nat. Sci.) 43(2) 363

RESULTS AND DISCUSSION 6845.40, 8114.43 and 1730.32 µmol TE/g, respectively (Table 1). The results from the ORAC Three extract samples of galangal were test showed a similar trend as previously described used in this study: water extract, ethanolic extract in the DPPH test. and essential oil. The water extract was a light Table 2 compares the effectiveness for brown powder that was almost odorless. The DPPH radical scavenging of galangal extracts with ethanolic extract was a light brown powder with a the commercial antioxidants: BHA and mild galangal aroma. The essential oil was a α-tocopherol. These two were the most effective colorless, clear liquid with a pungent and spicy antioxidants with IC50 = 0.41 and 1.45 mg/ml, odor. The strong odor of the essential oil could respectively. Among the galangal extracts, the limit its use to only specific foods in which the ethanolic extract was the most effective radical galangal aroma is acceptable. scavenger with IC50 = 10.5 mg/ml. From the IC50 data, the extract samples had lower antioxidant Antioxidant activities of galangal extracts activities than BHA and α-tocopherol. This was The antioxidant activities of the samples different from what was reported by Jitoe et al. were studied using the DPPH and ORAC methods. (1992), who found that the antioxidant activity of To obtain the ethanolic extract, 50% ethanol in an acetone extract of fresh galangal was higher water was used. It was the lowest concentration than that of α-tocopherol. This could have been of ethanol that produced the extract with high due to the use of a different solvent as well as antioxidant activity in the preliminary study. The differences in the raw material preparation. In this preliminary experiment showed that galangal study the galangal was dried before the extraction, extracts obtained by using 50 and 75% ethanol at but Jitoe et al. (1992) extracted it directly from 30°C and 50°C exhibited similarly high the fresh rhizomes. antioxidant activities at 33.58-36.63% inhibition. The extract that used 95% ethanol exhibited a Total phenolic compounds and total flavonoids lower percent inhibition at 11.41-14.41%. Phenolic compounds especially The data from the DPPH test showed that flavonoids including catechin are superoxide ethanolic extract had the highest antioxidant scavengers and show a strong antioxidant activity activity followed by the water extract and the (Hanasaki et al., 1994). In this experiment, essential oil (Figure 1). The antioxidant activities galangal extracts were analyzed for their total in all samples were concentration dependent. This phenolic compounds using Folin-Ciocalteu agreed with the work on galangal extracts and reagent (FCR) and total flavonoids were analyzed essential oil by Zaeoung et al. (2005). In the current by spectrophotometric assay. study, galangal extracts were also mixed with BHA The ethanolic extract had the to observe the effect when used together. The significantly highest total phenolic content, results in Figure 1 indicate that the mixture had whereas in the water extract and essential oil merely an add-on effect on antioxidant activity. samples this was not significantly different The ethanolic extract of galangal had the potential (p> 0.05). The total phenolic compounds in the to be used as an antioxidant and could partially water extract, ethanolic extract and essential oil replace BHA to reduce the use of BHA but it did were 8.25 ± 0.78, 31.49 ± 4.09 and 5.01 ± 0.14 not have a synergistic effect. mg GAE/g extract, respectively (Table 3). The ORAC values of the galangal water Antioxidant activity (ORAC method) was well extract, ethanolic extract and essential oil were correlated with total phenolic content (r = 0.800, 364 Kasetsart J. (Nat. Sci.) 43(2)

(a) 60

50 )

I 40

30

20 Inhibition (% Inhibition 0%BHA 10 0.01%BHA 0 0.0 1.0 2.0 3.0 4.0 5.0 Concentration of water extract (%,w/v)

70 (b) 60

) 50 I

40 on (%

ti 30

20

Inhibi 0%BHA 10 0.01%BHA 0 0.0 0.2 0.4 0.6 0.8 1.0 Concentration of ethanol extract (%,w/v)

50 (c)

40 ) I 30 on (%

ti 20

Inhibi 10 0%BHA 0.02%BHA 0 0.0 5.0 10.0 15.0 20.0 25.0

Concentration of essential oil (%, v/v)

Figure 1 Antioxidant activities at different concentrations, with and without BHA of (a) galangal water extract, (b) ethanolic extract and (c) essential oil. Kasetsart J. (Nat. Sci.) 43(2) 365

Table 1 The ORAC values of water extract, Table 2 The IC50 of BHA, α-tocopherol, water ethanolic extract and essential oil. and ethanolic extracts and essential oil Sample ORAC value 1/ of galangal. 1/ (µmol TE/g extract) Sample IC50 (mg/ml) BHA 0.41a Water extract 6845.40 ± 89.19 b α-Tocopherol 1.45 a Ethanolic extract 8114.43 ±718.48 c Water extract 54.39 c a Essential oil 1730.32 ± 80.73 Ethanolic extract 10.50 b 1/ Different letters within the same column indicate Essential oil 431.69 d significant differences between treatments based on one- 1/ Different letters within the same column indicate significant way ANOVA (p ≤ 0.05). differences between treatments based on one-way ANOVA (p ≤ 0.05). p ≤ 0.01). On a dry weight basis, the essential oil that some phenolic compounds in the essential oil fraction of galangal contributed much less phenolic probably belonged to groups other than flavonoids. compounds in comparison to the ethanolic and water extracts. HPLC analysis The amounts of total flavonoids in the The phenolic compounds in the samples extracts are shown in Table 3. The flavonoid were further analyzed using HPLC. The water and content of the water extract, ethanolic extract and ethanolic extracts were rich in phenolic essential oil was 1.48 ± 0.02, 13.78 ± 0.60 and compounds but the essential oil contained only a 0.20 ± 0.01 mgCE/g extract, respectively. The trace amount when analyzed with HPLC. antioxidant activity (ORAC method) of galangal The results in Table 4 and Figure 2 show extract correlated well with the flavonoid content that the water extract contained mainly myricetin (r = 0.720, p ≤ 0.05). (14.60 mg/g extract) and an unknown substance, Although the water extract and essential A (13.79 mgCE/g extract). The ethanolic extract oil contained approximately the same amount of had the unknown substance A (2.32 mgCE/g total phenolic compounds (Table 3), the essential extract) and catechin (1.74 mg/g extract) as its oil was lower in total flavonoids. This indicated major phenolic compounds. The extract also

Table 3 Total phenolic and flavonoid content of water extract, ethanolic extract and essential oil of galangal. Sample Concentration 1/ Total phenolics mg GAE/g extract mg GAE/g dry galangal Water extract 8.25 ± 0.78 a 2.94 ± 0.28 b Ethanolic extract 31.49 ± 4.09 b 10.28 ± 1.33 c Essential oil 5.01 ± 0.14 a 0.10 ± 0.00 a Total flavonoids mg CE/g extract mg CE/g dry galangal Water extract 1.48 ± 0.02 b 0.53 ± 0.08 b Ethanolic extract 13.78 ± 0.60 c 4.50 ± 1.74 c Essential oil 0.20 ± 0.01 a 0.004 ± 0.00 a 1/ Different letters within the same column of the same analysis indicate significant differences between treatments based on one-way ANOVA (p≤0.05). 366 Kasetsart J. (Nat. Sci.) 43(2) contained p-coumaric acid and two other unknown (Zaeoung et al., 2005), curcuminoids (Jitoe et al., substances. Several minor compounds in the 1992), p-hydroxy cinnamaldehyde and [di-(p- ethanolic extract were unidentified in this hydroxy-cis-styryl)] methane as reported in a study experiment (Table 4, Figure 3). These compounds on the chloroform extract of galangal rhizomes could have been p-coumaryl-9-methyl ether by Barik et al. (1987).

Table 4 Phenolic compounds in galangal extracts, analyzed by HPLC. compound Water extract Ethanolic extract mg/g extract mg/g dry galangal mg/g extract mg/g dry galangal Catechin 0.73 ± 0.29 0.26 ± 0.10 1.74 ± 0.26 0.57 ± 0.08 p-Coumaric acid - - 0.07 ± 0.00 0.02 ± 0.00 Myricetin 14. 60 ± 2.15 5.21 ± 0.77 0.45 ± 0.03 0.15 ± 0.01 Unknown A 1/ 13.79 ± 1.33 4.92 ± 0.47 2.32 ± 0.15 0.76 ± 0.05 Unknown B 1/ 0.47 ± 0.05 0.17 ± 0.02 1.35 ± 0.08 0.44 ± 0.03 Unknown C 1/ 0.06 ± 0.01 0.02 ± 0.00 1.52 ± 0.10 0.50 ± 0.03 1/ Concentrations of the unknowns are reported as relative concentrations to that of the catechin standard.

Figure 2 HPLC chromatogram of the water extract of galangal rhizomes.

mAU

25

20

15

10 catechin unknown A

5 unknown C unknown B -coumaric acid myricetin p

0 5 10 15 20 25 30 min

Figure 3 HPLC chromatogram of the ethanolic extract of galangal rhizomes. Kasetsart J. (Nat. Sci.) 43(2) 367

GC analysis 4-ol, α-terpineol, geranyl acetate, methyl eugenol The volatile phenolic compounds and β-caryophyllene. All of these compounds and their derivatives were analyzed by GC-MS were detected in the essential oil from this study (Table 5). It was clear that most phenolic (data not shown). compounds in the essential oil were volatile The water and ethanolic extracts components, being: chavicol, methyl eugenol, contained only small amounts of volatile thymol and eugenol. The major phenolic phenolics. However, there was a phenylpropanoid, compounds in the essential oil of galangal were namely 1´-acetoxychavicol acetate (ACA), which methyl eugenol (4130.38 µg/g extract) and could have contributed to the antioxidant activity chavicol (2390.45 µg/g extract). Essential oils of the ethanolic extract (Table 5). In this study, from other spices that showed high antioxidant ACA was present at a high concentration activity usually contained high eugenol (Tomaino (10 563.30 mg/g extract) and was found only in et al., 2005). The essential oil of galangal in this the volatile fraction of the ethanolic extract (Table study had a low concentration of eugenol and low 5). It was identified by matching the mass spectrum antioxidant activity. Most compounds in the (Figure 4) with that of ACA from galangal seed essential oil were terpenes, with Dorman et al. (Mitsui et al., 1976). ACA was also isolated from (1995) reporting that there were antioxidant galangal oleoresin extracted with pentane (Yang properties in: α-pinene, camphene, sabinene, and Eilerman, 1999) and from the dried galangal β-pinene, myrcene, ρ-cymene, 1,8-cineole, rhizome extracted with n-pentane/diethyl ether limonene, γ-terpinene, linalool, borneol, terpinene- (Janssen and Scheffer, 1985). The antioxidant

Table 5 Relative concentrations of volatile-phenolic compounds and phenylpropanoids in the extracts from rhizomes of galangal, analyzed by GC-MS. Compound RI ID 1/ Concentration HP-5 FFAP µg/g extract µg/g dry galangal Water extract Chavicol (4-allylphenol) 1259 2359 a, b, c1 21.24 ± 1.85 7.56 ± 0.66 Vanillin 1417 - a, b 2.92 ± 0.60 1.04 ± 0.21

Ethanolic extract Chavicol (4-allylphenol) 1260 2359 a, b, c1 257.39 ± 43.25 84.11 ± 14.13 Methyl eugenol 1411 2022 a, b, c1 87.63 ± 13.37 28.64 ± 4.37 Eugenol - 2195 a, b 13.32 ± 2.92 4.35 ± 0.95 1'-Acetoxychavicol acetate 1655 2505 c3 10 563.30 ± 3238.97 3452.06 ± 1058.49

Essential oil Thymol 1289 1903 a, b, c2 48.56 ± 7.16 1.00 ± 0.15 Chavicol (4-allylphenol) 1256 2350 a, b, c1 2390.45 ± 1387.35 49.28 ± 28.60 Methyl eugenol - 2022 a, b 4130.38 ± 139.07 85.16 ± 2.87 Eugenol - 2183 a, b 782.30 ± 36.65 16.13 ± 0.76 1/ ID = Identification by a = comparison of MS data with Wiley 275 library; b = comparison of RI with those in the ESO 2000 database of essential oil (Boelens Aroma Chemical Information Service, 1999)9; c = comparison of RI with literature data; c1 = Adams (1995); c2 = Lorjaroenphon (2004); c3 = Mitsui et al. (1976). 368 Kasetsart J. (Nat. Sci.) 43(2)

Abundance 132 3200000 150 3000000 2800000 2600000 2400000 192 2200000 2000000 1800000 1600000 1400000 1200000 43 1000000 121 800000 600000 77 103 400000 174 200000 31 55 65 94 165 234 0 40 60 80 100 120 140 160 180 200 220 240 m/z-->

Figure 4 Mass spectra and structure of 1´-acetoxychavicol acetate from the ethanolic extract of galangal rhizomes. activities of ACA and its related compounds that LITERATURE CITED formed during heating were studied by Kubota et al. (2001). Their data from ferric thiocyanate and Adams, R.P. 1995. Identification of Essential Oil TBA assays indicated that ACA had a higher Component by Gas Chromatography/Mass antioxidant activity than α-tocopherol and was Spectroscopy. Allured Publishing close to that of butylated hydroxytoluene (BHT). Corporation. Illinois USA. 469 p. In this study, ACA was one of the major peaks in Boelens Aroma Chemical Information Service. the GC-MS chromatogram of the ethanolic extract. 1999. ESO data base of essential oil. It could be extrapolated that the highest antioxidant Barik, B.R., A.B. Kundu and A.K. Dey. 1987. Two activities of the ethanolic extract were a result of phenolic constituents from Alpinia galangal its phenolic compounds and ACA. rhizome. Phytochemistry 26(7): 2126-2127. Carrubba, A. and I. Calabrese. 1998. Antioxidant CONCLUSIONS compound in some herbaceous aromatic plants. Acta Hort. 457: 85-93. The galangal ethanolic extract in this Cheah, P.B. and N.H. Abu Hasim. 2000. Natural study showed potential to be used as a natural antioxidant extract from galangal (Alpinia antioxidant in food products. Having phenolic galanga) for minced beef. J. Agric. Food compounds and ACA with a milder odor makes it Chem. 80(10):1565 – 1571. advantageous for use with more varieties of food Dorman, H.J.D., S.G. Deans and R.C. Noble. products when compared to other herbs and spices 1995. Evaluation in vitro of plant essential oil that have pungent terpenes as their antioxidative as natural antioxidant. J. Essent. Oil Res. 7: components. 645-651. Hanasaki, Y., S. Ogawa, and S. Fukui. 1994. The ACKNOWLEDGEMENT correlation between active oxygen scavenging and antioxidative effects of flavonoids. Free The authors would like to express their Radical Biol. Med. 16: 845-850. gratitude to the Graduate School, Kasetsart Itokawa, H., H. Morita, T. Sumitomo, N. Totsuka University for partially supporting this research. and K. Takeya. 1987. Antitumor principles Kasetsart J. (Nat. Sci.) 43(2) 369

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